This project continues my laboratory's studies of cellular actions of alpha1-adrenergic receptors using a clonal isolate of Madin Darby kidney (MDCK) cells. In recent studies our laboratory has shown that in these cells alpha1-adrenergic receptors may be of a unique subtype that links to a guanine nucleotide binding (G) protein and in turn to multiple phospholipases, including one or more phospholipases A2, C and D. Regulation of phospholipase A2 by alpha1-adrenergic receptors occurs in part via protein kinase C, in particular, by the alpha isoform of this enzyme. the current studies are designed to test hypotheses related to alpha-adrenergic receptor structure, phospholipase regulation, and protein kinase C isoforms in MDCK-D1 cells. Pharmacological methods will be used to define alpha1-receptor subtypes and subsequent molecular biological methods will be used to characterize the subtype(s) present in MDCK D1 cells. Studies of phospholipases will involve biochemical characterization of phospholipase D and phospholipase A2. The third aim is to assess the role of isoforms of protein kinase C in cell regulation, especially in regulation of phospholipase A2. The methods to be used in these latter studies include treatment of cells with antisense oligonucleotides and transient and stable transfection with antisense cDNA's to generate cells null with respect to individual C-kinase isoforms. Other approaches will involve microscopy with immunological probes for particular isoforms. Taken together, the studies proposed should provide new information regarding alpha1-adrenergic receptor action and the role of protein kinase C isoforms in regulation of cellular function. Given the importance of alpha1-adrenergic receptors and protein kinase C for a wide variety of cellular functions, the results may have importance to disease settings such as cancer and several types of cardiovascular disorders.